Superovulation is an efficient technique to obtain progeny from genetically valuable females. The ovarian response of each female depends on the number of gonado-sensitive follicles present at the time when treatment is initiated. Identification of the number of such follicles for each female would improve the efficacy of superovulation, by removing potential non-responders from the program. The AFC at a given point in time reflects the ovarian follicular reserve and has been positively correlated with superovulatory responses in donor cows [16, 10]. Dairy cows with a low number of ovarian follicles (≤ 15) have lower reproductive performance as compared to cows with a high number of follicles growing during follicular waves [17]. Considering the relationship between AFC and fertility characteristics, the donor selection could be performed using a single ultrasound examination at the beginning of superovulatory treatment [18]. Sahiwal donor of the present study responded positively to the gonadotropin treatment which resulted in the majority of the small follicle on the day of SO treatment being converted into a large size follicle pool on the day of SOE. This supports the hypothesis that gonadotropin treatment increases the number of antral follicles, favors the growth of small and medium follicles, and protects the small follicles (> 1.7 mm) undergoing atresia [8]. Further, it was recorded that the number of large follicles at SOE was positively correlated with the number of small follicles at the initiation of superovulatory treatment.
Results from this study indicate that pFSH treatment superinduces follicular growth in cows resulting in an increased proportion of small follicles growing into larger size classes. The increase in the numbers of medium and large antral follicles occurred in conjunction with an increase in the proportion of granulosa and thecal cells undergoing proliferation in these follicles [ 19, 20]. On the day of administration of PG (day 12) during superovulatory protocol, there is an increase in medium-size follicles (MF) (> 5mm), this finding is similar to that of [15, 21]. It occurs due to the rapid growth of the small follicle category to become medium in the presence of gonadotropin (pFSH) which supports the growth of the small follicles and prevent their atresia. Further, on the day of SOE (day 14) the higher number of large follicles (LF) was recorded due to the shift of MF to the LF category, whether or not they were destined to ovulate. Similar observations were recorded by other researchers [ 22, 23].
Atresia, the degeneration of ovarian follicles, occurs at all stages of follicular growth and development. However, at the early antral stage, follicles are most susceptible to atresia rather than in the preantral and preovulatory stages [ 9, 6]. The present study showed a significant decrease in the number of atretic follicles and atresia rate in the pFSH-stimulated group compared to the non-stimulated group. The administration of FSH also changes the balance between healthy and atretic follicles by preventing or delaying atresia [24, 25]. Thus, the increased numbers of follicles in the stimulated group and lesser atresia rate after pFSH administration in this study likely resulted in part from the continued growth of follicles that normally would have undergone atresia.
The number of small antral follicles of 3–6 mm in diameter in both the ovaries of the Sahiwal donor before superovulatory treatment was found to be significantly correlated with the number of large follicles at superovulatory estrus and ovarian size. Ovarian stimulation with a superovulatory dose of pFSH results in increased numbers of large growing follicles, of which approximately 75% or more were ovulated and ovulation is spread over 24 to 48 h, and a similar observation was made by [9]
After pFSH administration small follicles grow at a double growth rate (2.14 mm/day) to become medium size follicles, as compared to non-stimulated cows where follicle grows at 1.08mm/day and a similar type of pattern was recorded earlier where almost double follicular growth rates (3 mm/day) was seen during super stimulation [26]. However, In the present study, a significant 2 to 2.5 mm lesser preovulatory follicle size was recorded in superovulated Sahiwal donor cows than POF size in non-stimulated cows and earlier also similar findings were reported where 3 mm lesser preovulatory follicle size was observed in superovulated cows non-stimulated cows [9].
The serum progesterone at the initiation of super stimulatory treatment was slightly higher than reported by Sheetal et al. [27] and Prasad et al. [28] in Sahiwal cows. The mean concentration of P4 reached a basal level at SOE. Similar reports are shown by [29, 27] in Sahiwal cows. On the other hand, non-stimulated Sahiwal has a comparable concentration of P4 on the 10th, 12th, and 14th days of the estrus cycle. The decrease in the concentration of P4 on the day of SOE is due to the luteolytic dose of prostaglandin administered on the day 12th of superovulation.Mean plasma E2 concentration was unchanged in response to exogenous pFSH until after PG injection when it increased progressively by twofold in stimulated cows during the period between PG injection and mean expression of estrus compared to unstimulated. Similar findings were reported by Karl et al. [30]A significant rise in E2 was recorded on the day of superovulatory estrus in the present study, which was supported by Prasad et al. [28] and Siddiqui et al. [29] who also reported a higher level of E2 on the day of SOE as compared to respective days of the estrous cycle of non-stimulated Sahiwal cows. In mono-ovulatory, the higher concentration of E2 on the day of SOE might be due to the presence of a greater number of preovulatory follicles and capable of producing higher estradiol as compared to normal estrus, where usually one POF is present.
Estradiol-17β has various roles during follicular development and estrus and circulating estradiol concentrations are positively related to the intensity and duration of estrus [31]. In the present study, the serum estradiol level and estrus intensity were significantly (p < 0.05) higher in superstimulated Sahiwal cows than in normal cycling indicating the relationship between estrus behavior and serum estradiol concentrations. Superstimulated Sahiwal cows exhibited higher estrus intensity for a longer duration as compared to normal cyclic animals. Further, a majority of non-stimulated Sahiwal cows were of weak estrus intensity (50%), and similar observations were reported by Layek et al. [32]. A copious amount of discharge, vulvar hyperemia, moistening of the vulva, and uterine tonicity appeared early and evident in all the superstimulated Sahiwal donors with intense expression of various estrus characteristics. Further, the majority of the Sahiwal showed standing to be mounted and mounted on herd mates during SOE, which is in agreement with those reported by Negussie et al. [33] on Fogera cows (Bos indicus). However, these two estrus signs were expressed for a short period of time in the majority of the normal cycling Sahiwal cows [32].
The estrus duration in stimulated Sahiwal cow is in consonance with the earlier report of Khinda et al. [34], Barnes et al. [35], and Karl et al. [30], who also reported a similar estrus duration (30–54 hr) after gonadotropin treatment in cows. The estrus duration recorded in unstimulated Sahiwal was slightly higher than those reported by earlier workers (15.54 ± 0.28 h, Venkata Naidu and Rao [36] and 14.53 ± 0.80 h Layek et al. [32] in Sahiwal cattle) and by Pinheiro et al. [37] in Nelore cattle (10.5 ± 1.0 h). The reason for the difference in the duration between the studies might be due to different criteria, used to describe the onset and the end of estrus. The observation schedule followed by different workers may also be the reason for the difference.